AS-BandMEF-reprogramming

Analysis of the alternative splicing (AS) changes in B cell reprogramming, correlation of expression of RNA-binding proteins and comparison to MEF reprogramming. Published in Vivori et al., 2020.

00. Run vast-tools and edgeR and apply initial filtering

Datasets:

• B cell reprogramming dataset: Stadhouders et al., 2018.
• MEF reprogramming dataset: Cieply et al., 2016.
• List of RNA-binding proteins (RBPs) and splicing-associated RBPs: generated from Uniprot (RBP-SPL_Lists).

Alternative splicing analysis:

• Vast-tools v2.2.2 align (mm10), combine, compare with (00_Run_VTS.sh). See vast-tools webpage for details.
• Run vast-tools compare on all possible couples of samples for a big dataset with VTS_run_compare_all_couples.pl. Run perl VTS_run_compare_all_couples.pl to show help message.
• Filtering of vast-tools output for reads coverage with VTS_INCL_filtering.R, to consider only AS events with a minimum of 10 actual reads per sample (0 “N” values allowed for each dataset).
  • VTS_INCL_filtering.R filters vast-tools combine output (INCLUSION table) for reads coverage. A maximum number of “N” values (less than 10 actual reads) is allowed in the quality score columns of the selected samples (Score 1: Read Coverage, see vast-tools combine output format). Run Rscript --vanilla VTS_INCL_filtering.R -h to show help message.
    Outputs: two filtered INCLUSION tables (with and without quality scores).
• Extension of the filtered INCLUSION tables, including all the AS events differentially spliced in at least one comparison of each dataset (and their dPSI in all the comparisons).
  • VTS_add_dPSI_toINCL.R creates a union of vast-tools combine output (INCLUSION table) and multiple vast-tools compare outputs (DiffAS tables). It generates an extended version of the INCLUSION table, including all the events differentially spliced in at least one comparison (and their dPSI in all the comparisons). Run Rscript --vanilla VTS_add_dPSI_toINCL.R -h to show help message.
    Outputs:
    • If the input is the FILTERED INCLUSION table (see VTS_INCL_filtering.R), the output is a table with ALL CORRECTLY MAPPED events in the dataset (with dPSI from DiffAS files).
    • If the input is the original INCLUSION table, the output is a table with ALL events of the inclusion table (with dPSI from DiffAS files).

Gene Expression analysis (00_Run_edgeR.R):

• Import of gene counts (from STAR mapping)
• Filtering for minimum 5 cpms in at least in 33% of samples (5 for B cell reprogramming, 6 for MEF reprogramming)
• Calculation of cpm values and differential expression analysis.

01. Import and processing of AS and gene expression analyses outputs

• Import vast-tools outputs (01_Import_VTS.R).
• Import edgeR outputs (01_Import_edgeRcpm.R).

02. Analysis of AS events and gene expression of RNA-binding proteins

• Extract gene expression profiles from RNA-binding proteins and splicing factors (02_Extract_RBPscpm.R).
• Count and extract different types of AS events (02_VTS_TypeEvents.R).
• Melt dataframes for plots (02_mdfs_toplot.R).

03. Correlation of reprogramming stages, PCA analysis and other plots

• Correlation of B cell reprogramming stages according to AS or gene expression (03_Plot_CorrelationStages.R).

• Calculate and plot numbers of each type of AS events (stacked bar plot, heatmap) (03_Plot_StackedBarplot_VTSevents.R).

• Calculate overlap between AS events in B cell and MEF reprogramming & plot Venn diagram (03_Plot_VennDiagrams_BandMEFstoiPS.R).

• Principal component analysis (PCA) of B cell and MEF reprogramming on most variable genes & generate plot (03_PCA_BandMEFstoiPS.R).

• Plot heatmaps of scaled PSI values of differential cassette exons (03_Plot_Heatmap_VTS_CEx_BandMEFstoiPS.R).

• Plot heatmap of scaled expression of RNA-binding proteins involved in pluripotency (data from Han et al., 2013), reprogramming or development (03_Plot_Heatmap_RBPs_HanDev_BandMEFstoiPS.R).

• Plot expression of genes in B cell and MEF reprogramming according to the PCA-derived 'reprogramming pseudotime' (03_Plot_Expression_BandMEFstoiPS.R).

04. Clustering of AS exons in B cell reprogramming and correlation of RBPs expression

• Mfuzz clustering of differentially spliced cassette exons (04_Clustering_VTS_CEx.R).
• Correlation of gene expression profiles of RBPs to each AS cluster (04_Clusters_Assign_RBPs_GEx.R).

05. Analyses on AS clusters

• Expression of genes containing the AS exons belonging to each AS cluster (05_Clusters_Assign_GEx_VTS.R).
• Prediction of ORF disruption effects for exons in AS clusters (05_ORFdisruption_Clusters_CEx.R).
• Calculate overlap between AS clusters in Bcell reprogramming and MEF reprogramming (05_Overlap_VTS_BandMEFstoiPS.R).